OP-22 - Bicarbonate is required for hydrogen peroxide-dependent inactivation of PTP1B in presence of the Trx system and peroxiredoxin

Abstract

Protein tyrosine phosphatases need to be inhibited by oxidation of their active site cysteine during growth factor signaling. It is not clear how this inhibition is mediated but bicarbonate (HCO3-) has been shown to potentiate PTP1B inactivation. Therefore we investigated the effects of HCO3- and H2O2 on PTP1B activity in the presence of a reducing system constituted of Prx2, Trx1 and TrxR1. Recombinant PTP1B, TrxR1, Trx1 and Prx2 together with NADPH were used to mimic cellular growth factor-dependent PTP1B regulation in vitro. Effects of HCO3- and H2O2 exposure were assessed by PTP1B activity analysis. We found that HCO3-- and H2O2 inactivated PTP1B also in the presence of the complete TrxR1 / Trx1 / Prx system. Interestingly, a dynamic PTP1B inactivation pattern mimicking an oxidative burst was observed. The capacity for PTP1B reactivation was proportional to either TrxR1 or Trx1 concentrations. In summary, HCO3- and H2O2 in combination potently negatively regulates PTP1B activity. TrxR1 was previously shown to prevent H2O2 dependent PTP1B inactivation. Here we found that HCO3- with H2O2 together potently overcomes TrxR1- and Trx1 -mediated protection of PTP1B and that its subsequent reactivation is dependent upon Prx2, where H2O2 clearance becomes a key regulatory event.